Our long-term goal is to uncover molecular mechanisms that contribute to the increased incidence of hepatocellular carcinoma (HCC) in the US. In order to understand potential molecular mechanisms that may drive HCC development and progression, we performed whole genome RNA sequencing using total RNA samples from paired adjacent non-tumor liver and HCC tumor tissues of local HCC patients. Analysis of the differentially expressed genes between the paired tissues revealed significant alterations of Biological Processes and Molecular Pathways associated with oxidation reduction, which involves a gene coding for Six Transmembrane Epithelial Antigen of Prostate 2 (STEAP2) protein. We found that STEAP2 was also significantly upregulated in the tumors in comparison to adjacent non-tumor liver tissues of the patients in The Cancer Genome Atlas (TCGA) dataset as well as in two recently published RNA-seq datasets. STEAP2 belongs to a family of proteins involved in the reduction and transport of iron and copper ions across membranes of a cell and various cellular organelles. The reduced forms of iron and copper ions are not only essential elements needed for uncontrolled tumor cell growth, but also known to mediate the production of hydroxyl radicals from hydrogen peroxide, which cause DNA and protein damage and lipid peroxidation. Consistent with these functions of STEAP2, we found total levels of copper were significantly higher in the HCC tissues than in the adjacent non-tumor tissues. Knockdown of STEAP2 expression in human HCC cell lines significantly inhibited their viability, motility, clonogenicity in soft agar, and xenograft growth in vivo along with decreased stress-activated MAP kinase activity and intracellular iron and copper levels, whereas STEAP2 overexpression showed opposite effects. Furthermore, a high iron diet significantly increased HCC incidence in a mouse model. Although hepatic copper or iron overload associated with Wilson's disease and hemochromatosis respectively is a known risk factor for HCC, whether dysregulation of copper and iron homeostasis due to STEAP2 overexpression may contribute to hepatocarcinogenesis has not been explored. On the basis of our novel preliminary findings, we hypothesize that STEAP2 upregulation can drive HCC development and progression via increased supply of ferrous and cuprous ions, oxidative stress, and lipid peroxidation resulting in the activation of stress-activated pathways. In specific aim 1, we will use newly established patient-derived HCC organoid cultures and a spontaneous mouse model of HCC to determine whether altered expression of STEAP2 will affect their malignant properties in vitro and in vivo. In specific aim 2, we will first determine whether STEAP2 possesses metalloreductase catalytic activity and whether its N- terminal domain has a well-defined NADPH binding site and its C-terminal domain binds heme in HCC cells. We will then engineer site-directed mutations of STEAP2 to determine whether its catalytic activity is necessary for its tumor-promoting activity including cell growth and migration, and tumorigenic property in HCC cells. In specific aim 3, we will determine whether altered expression of STEAP2 results in increased labile iron and copper, oxidative stress, DNA damage, and protein adduct formation in HCC cells. We will investigate whether STEAP2-stimulated oncogenic pathways is dependent on iron and copper accumulation. We will also determine whether stress-activated MAP kinases mediate the HCC-promoting activities of STEAP2. We are the first to show the upregulation and tumor-promoting functions of STEAP2 in HCC. Given its role in promoting cancer cell growth and intracellular iron/copper accumulation leading to oxidative stress and activation of oncogenic pathways, our proposed research promises to uncover a novel molecular mechanism contributing to hepatocarcinogenesis and to reveal actionable targets for the development of novel preventive and therapeutic strategies.